Double stranded oligodeoxynucleotides (ODN or “decoys”) for reducing trans-activity of transcription factors are an innovative and attractive strategy for gene therapy and for the functional study of gene products. Several different double-stranded DNA structures, including unmodified oligonucleotide duplexes, ab-anomeric oligonucleotides, phosphorothioate oligonucleotide duplexes, and dumbbell oligonucleotides, have been introduced as decoys for transcription factors (see Scholer H R and Gruss P., Cell 1984; 36: 403-411; Cereghini Set al. Genes Dev 1988; 2: 957-974; Berkowitz L A. et al., Mol Cell Biol 1989; 9: 4272-4281; Tanaka N as al., Nucleic Acids Res 1994; 22: 3069-3074; Bielinska A et al., Science 1990; 750:997-1000; Clusel C at al., Nucleic Acids Res 1993; 21: 3405-3411; Lim C S at al., Nucleic Acids Res 1997; 25: 575-581; Hosoya T et. al., FEBS Lett 1999; 461: 136-140; Mann M J and Dzau. V J J. Clin. Invest. 2000; 106:1071-1075).
The transfection of double-stranded cis element decoy ODNs results in the sequestration of trans-activating factors from endogenous cis-elements of the same sequence, with subsequent inhibition of gene expression (see Bielinska A, as al., 1990 supra; Morishita R, at al. 1998 supra; and Sawa Y, Morishita R, Suzuki K., Circulation 1997; 96:II-280-II-285).
Moreover, the administration of antisense or decoy oligonucleotides against NF-κB, c-myb, c-myc, cdc2, cdk2, E2F, and CRE, has been shown to decrease cachexia (Kawamura I et al, Gene Ther. 1999;6:91-97), in vitro cell proliferation and intimal thickening in experimental restenosis (see Simons M et al., Nature 1992; 359: 67-73; Morishita R at al., J Clin Invest 1994; 93:1458-1464; Morishita R et al., Proc Natl Acad Sci USA 1993; 90: 8474-8478; Morishita R et al., 1995 supra; Morishita R at al., Nat Med 1997; 3: 894-899; Kaneda Y and Morishita R, Jpn J Clin Pathol 1997;45:99-105; Tomita et al. Am. J. Physiol. 1998;275:F278-F284; Ma shima Y et al, J. Clin. Invest. 1998; 101:2589-2597; Akimoto M et al., Exp Eye Res. 1998;67:395-401; Mann M J at al., Lancet, 1999;354:1493-1498; Mann and Dzau 2000 supra,: Kawauchi M et al, Circ. Res. 2000;87:1063-1068; Mangi A A and Dzau V J, Ann Med 2001;33:153-155; Ehsan A et al, J Thorac Cardiovasc Surg 2001;121:714-722; Kawauchi M at al. Transplant. Proc. 2001;33:451; McCarthy M, Lancet, 2001;358:1703), suppress proliferative cholangitis (Yoshida M et al, J. Surg. Res. 2002;102:95-101) and slow tumor growth and induce apoptosis in cancer models (Park Y G et al., J. Biol. Chem. 1999;274:1573-80; Cho-Chung Y S et al, Mol. Cell. Biochem. 2000;212:29-34; Alper O et al, Mol. Cell. Biochem. 2001:218:55-63), respectively.
The main limitation of unmodified oligonucleotide ODNs is that they are easily degraded by nucleases present in serum and in cells. In order to solve this problem, oligonucleotides with modified linkages such as phosphorothioate and methylphosphonate have been developed. However, these modified ODNs exhibit problems such as insensitivity to RNase H, the possibility of recycling of hydrolyzed modified nucleotides into cellular DNA, lack of sequence-specific binding effects of ODN-based gene therapy, and immune activation (see Moon I J, et al., J Biol Chem. 2000;275:4647-4653; Hosoya T, et al, FEBS Letters 1999;461:136-140; Khaled Z, et al., Nucleic Acids Res 1996; 24: 737-775; Gao W Y et al., Mol Pharmacol 1992; 41: 223-229; Brown D A et al., J Biol Chem 1994; 269: 26801-26805; and Burgess T L et al., Proc Natl Acad Sci USA 1995; 92: 4051-4055).
Recently, decoys have been proposed for treatment of diseases and disorders related to transcriptional factors including neointima formation. Neointima formation results from excessive proliferation and migration of vascular smooth muscle cells (VSMC) from media to intima, which are critical steps in the pathogenesis of atherosclerosis and restenosis which are the major problems following percutaneous transluminal coronary angioplasty (PTCA) (see Currier J W, and Faxon D P., J Am. Coll Cardiol 1995;25:516-520; Clowes A W, at al.; Lab Invest 1983;49:208-215; Liu M W, et al., Circulation 1989;79:1374-1387; Ross R., Nature. 1990;362:801-809; and Paulette P, et al., Clin. Sci. 1994;87:467-479).
There have been a number of trials with pharmacological agents to reduce the incidence and rate of restenosis after PICA, but the results have not been satisfactory. Over the last decade, anti-gene therapy, focusing on the inhibition of VSMC proliferation, has emerged as a potentially attractive strategy for reducing restenosis after PTCA (see Simons M, et al., Nature 1992;359:67-73; Morishita R, et al., J Clin Invest 1994;93:1458-1464; Morishita R, et al., Proc Natl Acad Sci USA 1993;90:8474-8478; Morishita. R, et al, Proc Natl Acad Sci USA 1995;92:5855-5859; Morishita R, et al., Nat Med 1997;3:894-899; Morishita R et al, Pharm. Ther. 2001;91:105-114, Motokuni A at al., Nippon Rinsho 2001;59:43-52).
Previous studies have found that extracellular signal-regulated kinase (ARK) and c-Jun NH2-terminal kinase (JNK), both belonging to the mitogen-activated protein kinase (MAPK) family, are rapidly and transiently activated after balloon-injury (see Ohashi N, et al., Arterioscler Thromb Vase Biol. 2000;20:2521-252:6; Koyama H, et al., Circ Res. 1998;82:713-721; Hu Y, et al., Arterioscler Thromb Vase Biol. 1997;17:2808-2816; and Pyles J M, et al., Circ Res. 1997;81:904-910).
ERK2 and JNK1 activities in the injured vessel wall rapidly increase after balloon injury and reach a high level at 5 minutes after injury. A sustained increase in ERK2 kinase activity was observed over the next 7 days in the arterial wall and 14 days in neointima after injury (Hu Y, et al, 1997 supra; and Izumi Y, et al., Circ Res. 2001:88:1120-1126).
JNK and ERK are known to be translocated into the nucleus, and activate c-Jun and c-Fos, which dimerize to form the transcription factor complex AP-1. AP-1 binds to specific DNA sequences present in a large number of genes associated with a diverse range of cell proliferative responses such as extracellular matrix production (see Karin M., J Biol Chem. 1995;270:16483-16486; and Whitmarch J. and Davis R J., J Mol Med. 1996;74:589-607), apoptosis (Le-Niculescu H et al., Mol. Cell. Biol. 1999;19:751-763; Taimor G. et al., FASEB J. 2001;15:2518-2520), vascular remodeling (Morishita R, et al., Biochem Biophys Res Common 1998;243:361-367; Lauth M. et al., J Mol Med 2000;78:441-450; Wagner A H et al., Mol. Pharm. 2000;58:1333-1340; Cattaruzza M. et al., J. Biol. Chem. 2001;276:36999-37003), COX-2 mediated inflammation (Adderley, S R and Fitzgerald D J, J. Biol. Chem 1999;274:5038-5046; von Knethen A et al., Mol. Biol. Cell 1999;10:361-372; von Knethen A et al., J. Immunology 1999;163:2858-2866; Subbaramaiah K et al., J. Biol. Chem. 2001;276:12449-12448) and production of type 1 plasminogen activator inhibitor (PAI-1) (Ann J D., et al., Diabetologia 2001;44:713-720) TGF-0 (Sin G and Howe P H, J. Biol. Chem. 1997;272:26620-26626) and IL-6 (Viedt C et al., FASEB J 2000;14:2370-2372).
These results suggest that AP-1 binding may be involved in vascular smooth muscle cell proliferation in response to vascular injury. However, it is not known whether inhibition of AP-1 binding would prevent neointima formation.
Recent reports have also shown that the transcription factor E2F, which forms a complex with cyclin A, cdk2, and pRB, and activates and phosphorylates these cell cycle regulatory genes, is critical to the process of cell growth and proliferation (see Pagano N at al. EMBO J 1992; 11: 961-971; Pardee A B. Proc Natl Acad. Sci USA 1974; 71: 1286-1290; Weintraub S J, et al. Nature 1992; 358: 259-261; Pagano M G, at al. Science 1992; 255: 1144-1147; and Rosenblatt J, at al. Proc Natl Acad Sci USA 1992; 89: 2824-2828).
The transcriptional factor nuclear factor-κB (NFκB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes that might be involved in myocardial damage after ischemia and reperfusion. Decoys specific for NFkB has been used in vivo to bind the transcriptional factor and to block the activation of genes mediating myocardial infarction, thus providing effective therapy for myocardial infarction (Morishita R. et al., Nat Med 1997 August; 3(8):894-9).
Although there have been successful application of decoys to some diseases and disorders related to transcriptional factors, the above-mentioned main limitation of unmodified oligonucleotide ODNs where they are easily degraded by nucleases present in serum and in cells, significantly reduces the efficacy of decoys in treatment and prevention of such diseases and disorders.
In in vitro studies, covalently closed ODNs have been developed to avoid exonuclease activities by enzymatically ligating two identical molecules, in order to overcome these limitations. Circular dumbbell oligonucleotides, which are made by the circularization of the oligonucleotides by joining the 3′ and the 5′ ends with enzymatic ligations, and have a non-toxic unmodified backbone which resembles natural DNA, have increased stability to exonucleases, had have increased uptake into cells as compared with the chemically modified linear oligonucleotides (See Chu B C F and Orgal L., Nucleic. Acids Res. 1992; 20:5857-5858; and Abe T, et al., FEBS Lett. 1998; 425:91-96).
However, there have been no reports showing that such covalently closed ODNs or circular dumbbell oligonucleotides are effective in treating or preventing diseases or disorders.